Myotis Lucifugus)

Habitat quality, torpor expression and pathogen transmission

in little brown bats (Myotis lucifugus)

Ana M. Breit

A thesis submitted to the Faculty of Graduate Studies

in partial fulfilment of the requirements for the

Master of Science Degree

Department of Biology

Master of Science in Bioscience, Technology, and Public Policy

University of Winnipeg

Winnipeg, Manitoba, Canada

March 26, 2018

Table of Contents List of Tables ...... 3 List of Figures ...... 4 CHAPTER 1: GENERAL INTRODUCTION ...... 5 Objectives and Hypotheses ...... 16 Ethical statement ...... 18 References ...... 19 CHAPTER 2: EFFECTS OF ROOST TEMPERATURE ON TORPOR EXPRESSION AND PATHOGEN ACQUISITION IN SOLITARY LITTLE BROWN BATS...... 29 Abstract ...... 29 Introduction ...... 30 Methods ...... 35 Results ...... 43 Discussion ...... 52 References ...... 57 CHAPTER 3: EFFECTS OF ROOST TEMPERATURE ON AGGREGATION AND TORPOR EXPRESSION BY GROUPS OF LITTLE BROWN BATS ...... 64 Abstract ...... 64 Introduction ...... 65 Methods ...... 71 Results ...... 80 Discussion ...... 96 References ...... 103 CHAPTER 4: GENERAL CONCLUSIONS...... 111

Acknowledgements I cannot overstate my gratitude to the numerous humans who helped me with this thesis. Thank you to my incredible supervisor, Craig Willis, who not only trained me as a researcher and writer, but also mentored me and literally gave me a bed to sleep on, as well as a few beers over the years. Thank you to Quinn “Heather Mayberry” Fletcher, for teaching me how to speak the language of R. You kept me sane and there would have been so many more tears and broken windows/computers if it hadn’t been for your patience and expertise. I owe you a lifetime of beers. A million and one thanks to Kaleigh Norquay. You were my first friend in Canada, and then my first family here too. Thank you so much for not only being my bat-sister and mentoring me in science, but even more for making me part of your family. You have an amazing support system that you shared with me and I cannot express my gratitude enough. Moving to Winnipeg was one of the hardest things I’ve done in my life, and thanks to you, moving away will be even harder. Thank you also to my amazing labmates and field technicians who literally kept me and my project alive. Specific thanks to Emma Kunkel, who stuck by me through not only the fieldwork mishaps but also through the analysis and writing portion of this thesis. Your companionship over the last couple years has meant more to me than words can describe (see Appendix 2: Love letter to Emma L. Kunkel). Thank you also to Yvonne Dzal, Nicole Dorville, Andrew Habrich, and Trevor Moore, for listening to so many renditions of this thesis and presentations, and then still being my friend afterwards. Working with you all has been amazing and I will miss you so much. Thank you also to my support system of fellow graduate students at the University of Winnipeg. It has been a fantastic ride with all of you. Finally, thank you to my family. Thank you, Yusuf, for not only the food, shelter, and protection, but also for the support, encouragement, and companionship. Your patience and humor kept this thesis a part of my life instead of the definition of my life. Thank you. I love you. And most importantly, thank you to my mom and dad. Thank you Papa, for taking me to into the woods before I could walk. Those memories of us listening to Sandhill cranes at our spot in the wedge are some of the happiest and most peaceful moments of my life. I love you. And thank you to my mom, for being so encouraging at times it was scary. Your love and enthusiasm is out of this world. Thank you especially for watching bats with me in the backyard, where it all began. I love you both so much. Thank you.

List of Tables

Table 2.1: Summary of morphometric data and raw data for each animal used in analysis of torpor expression and infection intensity. *Original value of zero was assumed to be below the limit of detection so was changed to the lowest detection divided by 2 (see methods)…………………………………………………………….....44

Table 2.2: Summary of linear mixed effects model assessing effects of average Troost (with trial as a random effect, p<0.0001) on normothermy index of 15 little brown bats (n=6 male, 9 female) over 9 24-hour trials………...………………………..45

Table 2.3: Summary of linear mixed effects model assessing effects of normothermy index (with trial as a random effect, p=0.0001) on pathogen acquisition of 15 little brown bats (n=6 male, 9 female) over 9 24-hour trials………………………….48

Table 3.1: Summary of linear mixed effects model assessing effects of group size, average Troost, their interaction term, sex, and mass (with trial as a random effect, p<0.0001) on normothermy index of 68 little brown bats (n= 22 male, 45 female) over 11 24-hour trials…………………………………………………………....85

Table 3.2: Summary of linear mixed effects model assessing effects of average Troost, sex, and mass (with trial as a random effect, p<0.0001) on normothermy index of 18 little brown bats (n= 8 males, 10 females) that roosted in small-sized groups (1-3 individuals)…………………………………………………………………..…..88

Table 3.3: Summary of linear mixed effects model assessing effects of average Troost, sex, and mass (with trial as a random effect, p<0.0001) on normothermy index of 35 little brown bats (n= 7 males, 28 females) that roosted in large-sized groups (7-10 individuals)………………………………………………………………………89

Table 3.4: Summary of linear mixed effects model assessing effects of average Troost, sex, and mass (with trial number as a random effect, p<0.0001) on nomothermy index of 14 little brown bats (n= 7 male, 7 female) that roosted in medium-sized groups (4-6 individuals)…………………….……………………………………………90

Table 3.5: Summary of linear mixed effects model assessing effects of group size, normothermy index, their interaction term, and sex (with trial as a random effect) on the logit-transformed intensity of infection of 68 little brown bats over 11 24- hour trails………………………………………………………………….……..92

List of Figures

Figure 2.1: Boxplot of daily average Troost in both heated and control roost boxes in heated and control flight enclosures. Centre lines represent median values, boxes represent the quartile range, the upper whisker represents the third quartile plus 1.5 times the interquartile range, and the lower whisker represents the first quartile minus 1.5 times the interquartile range. Open circles represent outliers..39

Figure 2.2 Representative time courses of Tsk (°C) (circles), Troost (°C) (black solid line), and Ta (°C) (gray solid line) for (A) one bat in the control enclosure and (B) one bat in the heated enclosure during one of my 24-hour trials on 18-19 August 2016………………………………………………………………………………46

Figure 2.3: Scatter plot of the relationship between daily average Troost (°C) and normothermy index for 15 little brown bats in heated (black circles) and control (gray circles) flight enclosures. Data points are coloured by Troost treatments for illustration but heated vs. control treatment was not included in the model. A linear regression line is also included for illustration although data were analysed using a mixed effects model (see results)………………………………………..47

Figure 2.4: Histograms of (A) un-transformed intensity of infection (i.e., proportion of the wing covered with UV powder), and (B) logit-transformed intensity of infection for 15 little brown bats during environmental-substrate transmission infection experiment……………………………………………………………..50

Figure 2.5: Scatter plot of the relationship between normothermy index and intensity of infection (proportion of wing covered by proxy pathogen) for 15 little brown bats in heated (black circles) and control (gray circles) flight enclosures. Data points are coloured by Troost treatments for illustration but heated vs. control treatment was not included in the model (see results)……………………………………...51

Figure 3.1: Boxplots of daily average Troost in both artificially heated and control roost boxes over the course of 11 24-hour trials (p<0.0001). Centre lines represent median values, boxes represent the quartile range, the upper whiskers represent the third quartile plus 1.5 times the interquartile range, and the lower whiskers represent the first quartile minus 1.5 times the interquartile range. Open circles represent outliers…………………………………………………………………75

Figure 3.2: Scatter plot of the relationship between average daily Ta (°C) and average daily Troost (°C). Open circles represent heated roosts and black circles represent control roosts. Linear regression lines are also included for illustration although data were analyzed linear mixed effects models including trial as a random effect (see methods)…………………………………………………………………….82

Figure 3.3: Proportions of bats roosting in one heated versus three un-heated control roost boxes over the course of 11 24-h trials…………………………………….82

Figure 3.4: Scatter plot of the relationship between the proportion of bats in a roost and daily average Troost (°C). A linear regression line is also included for illustration although data were analyzed using a generalized linear model (family=binomial, see results)……………………………………...... 83

Figure 3.5: Histogram of the group size (number of bats in roost) in which each of the 68 little brown bats chose to roost over 11 24-hour trials (see results)……………..86

Figure 3.6: Scatter plot showing the effect of average Troost on normothermy index. Group size showed a tri-modal pattern (see Figure 3.4) and is represented here by black circles for groups of 1-4 individuals, dark gray circles for medium-sized groups of 4-7 individuals, and white circles for large groups of 8-10 individuals. Relationships between groups size and NI were significant for small and large but not medium-sized groups (see results)…………………………………………...87

Figure: 3.7: Histograms of (A) un-transformed intensities of infection (i.e., proportion of the wing covered with UV powder), and (B) Histogram of logit-transformed intensity of infection showing normal distribution………………………………93

Figure 3.8: Scatter plot of the relationship between logit-transformed intensity of infection for each individual and the size of the group in which the bat was found (p=0.80)…………………………………………………………………………..94

Figure 3.9: Scatter plot of the relationship between logit-transformed intensity of infection for each individual and it’s NI (p=0.68)……………………………….95

CHAPTER 1: GENERAL INTRODUCTION

Emerging infectious diseases of wildlife are a growing concern for conservation as human travel and trade increase movement of pathogens, and habitat loss or fragmentation affect host-pathogen dynamics (Daszak et al. 2000, Wilder et al. 2015).

Introduced pathogens can have devastating effects on naïve host populations, in some cases driving host species to extinction and reducing overall biodiversity (Daszak et al.

2000, Smith et al. 2006, Mitchell et al. 2008). The impacts of a pathogen on a host population is a function both of pathogen traits (e.g., the basic reproductive number, R0,

Brunham et al. 1993), host physiology and behaviour (e.g., huddling) and the shared host-pathogen environment (e.g., resource patches like watering holes or oases, Anderson

& May 1979, Scholthof 2007). Most host-pathogen systems are thought to be dependent on host density such that as a host population declines pathogen transmission will similarly decline (Anderson & May 1992). However, some pathogens may also persist independent of host density, as long at the frequency of interactions between conspecifics is still high (Anderson & May 1992). Extinction caused by a pathogen is more likely when transmission is frequency-dependent, rather than density-dependent, as is the case for many sexually transmitted pathogens or pathogens that can survive and, especially, reproduce in the environment without hosts present (de Castro & Bolker 2005).

Environmental factors can exert indirect and direct effects on host physiology and behaviour in ways that can affect host susceptibility and disease impacts. For some host- pathogen systems, weather conditions and temperature may affect susceptibility by compromising immune function (Raffel et al. 2006). For example, when exposed to low ambient temperatures (Ta) of about 5°C for extended periods (2-5 months), leopard frogs

(Rana pipiens) show decreased proliferation of both T-cell and B- cell lymphocytes, which normally allow animal immune systems to identify antigens and mount an adaptive immune response (Maniero & Carey 1997). The reduction in immune response resulting from environmental variation increases susceptibility of frogs to Batrachochytrium dendrobatidis, the fungal pathogen that causes the devastating disease chytridiomycosis

(Raffel et al. 2006). Environmental variation can also affect pathogens in ways that might affect transmission. For example, B. dendrobatidis thrives in temperatures between 17° and 25°C but shows reduced growth and mortality above 28°C (Longcore et al. 1999).

The environment can also affect host behaviours. Chronic wasting disease, a prion disease affecting mule deer (Odocoileus hemionus, Miller et al. 2004), white-tailed deer

(Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelson, Miller et al.

1998) is transmissible from an infected individual’s saliva or feces to a susceptible ungulate (Mathiason et al. 2009, Tamguney et al. 2009). In areas where habitat quality is poor, large groups of cervids may aggregate in and around food patches which increases the likelihood of pathogen transmission both from infected individuals and substrates

(Joly et al. 2006,Almberg et al. 2011). An understanding of the importance of host behaviour and physiology, and the importance of the environment for host-pathogen interactions can be crucial for management of wildlife disease (Scholthof 2007). For example, aggregation at supplemental feeding areas is suspected to have contributed to the increased transmission of tuberculosis in deer (Palmer et al. 2004), chronic wasting disease in deer and elk (Cervus canadensis, Williams et al. 2002), and brucellosis in elk and bison (Bison bison, Etter & Drew 2006). By discouraging and outlawing artificial feeders, wildlife managers were able to manipulate behaviour of host species and reduce

8 aggregation, thus reducing the risk of pathogen transmission and acquisition (Brown &

Cooper 2006). Management strategies that are informed by host social behaviour and physiology can, therefore, be important for reducing impacts of infectious disease on wildlife populations.

A range of other host characteristics are also thought to influence host-pathogen dynamics, host susceptibility and disease impacts (Scholthof 2007). The density of host populations and aggregations, the lifespan/longevity of host individuals and sociality can all affect transmission throughout the population (Anderson & May 1992). For example, in Cape ground squirrels (Xerus inauris), females have smaller home ranges (Waterman

1995) and carry greater endoparasite loads than males, likely because they of their consistent exposure to conspecifics’ fecal pellets in foraging areas (Hillegass et al. 2008).

Males, who forage over large areas and nest in vacant burrows (Waterman 1995), are less likely to come in contact with infected feces (Hillegass et al. 2008). While a large body of theoretical research and epidemiological modeling exists (May & Anderson 1978,

Anderson & May 1979, Barlow 1991, de Jong et al. 1995, Roberts 1996, McCallum et al.

2001), relatively few empirical studies have addressed how variation in host behaviour might affect pathogen transmission and proliferation within a population, and fewer still have conducted controlled experiments on the influence of host behaviour on host- pathogen dynamics.

Energy limitation caused by environmental constraints could force animals to shift their behaviour in ways that affect host-pathogen dynamics. Endothermic animals must expend energy to maintain body temperature (Tb) during cold exposure which is particularly challenging during periods of low food abundance (Lovegrove et al. 2001,

Mzilikazi & Lovegrove 2002, Geiser 2004). As a result, some endothermic species huddle with conspecifics during inclement weather (e.g., striped skunks, Mephitis mephitis, Gunson & Bjorge 1979), Siberian flying squirrels (Pteromys volans, Selonen et al. 2014), yellow-eyed penguins (Megadyptes antipodes, Seddon & Davis 1989), and white-backed mousebirds (Colius colius, McKechnie & Lovegrove 2001), which could increase their risk of pathogen transmission and acquisition.

Some endothermic animals are capable of heterothermy, or torpor, a state of reduced metabolic rate and Tb which conserves energy during periods of harsh weather conditions and low food availability. Many species of birds and mammals are heterothermic endotherms that can selectively enter torpor or longer-term hibernation to save energy (Willis et al. 2006; Canale & Henry 2011). In Richardson’s ground squirrels

(Urocitellus richardsonii), for example, hibernating for an 8-month winter can reduce energetic costs by nearly 90% compared to remaining normothermic (Wang 1979). In addition to energy savings, torpor may also help individuals avoid predation by minimizing the amount of time spent foraging and exposed to potential predators

(Stawski & Geiser 2010). Subtropical insectivorous bats (Nyctophilis bifax) in good body condition enter torpor more often than individuals in poor condition, even during relatively high Ta and when food is abundant, possibly to reduce foraging requirements and avoid the risk of predation while foraging (Stawski & Geiser 2010). Predation avoidance as a side benefit of torpor expression suggests the additional possibility that increased use of torpor, and a concomitant reduction in activity, could also reduce the risk of acquiring pathogens and parasites. However, this potential indirect benefit of torpor has never been tested.

Although torpor expression reduces energetic costs and provides a number of additional benefits (Geiser & Brigham 2012), it also comes with associated costs

(Humphries et al. 2003). The largest and best studied of these potential costs is delayed reproduction and offspring growth, and reproductive mammals tend to avoid torpor expression compared to non-reproductive conspecifics (Geiser 1996, Lausen & Barclay

2003, Dzal & Brigham 2013). Torpor delays parturition for pregnant females, and inhibits lactation, both of which may be especially detrimental for hibernators as offspring will have less time to grow and build fat stores for their first winter (Geiser 1996). Torpor also delays sperm production for males (Jagiello et al. 1992) and can slow healing rates from injury (Faure et al. 2009). To avoid these costs of torpor, in some situations and at certain times of year, many heterothermic endotherms will select warm microclimates, or huddle with conspecifics to avoid torpor and its associated costs.

Another host behaviour that can affect host-pathogen dynamics is sociality.

Sociality, or the tendency of a species to live communally, has evolved in response to a suite of evolutionary pressures. Benefits of sociality include territorial control of resources (e.g., African lions, Panthera leo; Mosser & Packer 2009), protection from predation (e.g., meerkats, Suricata suricatta; Townsend et al. 2011), and ease of finding a mate (Hamilton, 1970). On the other hand, sociality can also be costly if it enhances the risk of acquiring pathogens or parasites (Altizer, et al. 2003). Highly social or colonial species are likely to have high contact rates with conspecifics and increase the potential for pathogen or parasite transmission such that less social individuals may benefit by avoiding group living (Alexander 1974). If pathogen risk is greater in large social groups,

11 there could be evolutionary pressure on group size, social interactions, and group composition (Côté & Poulin 1995, Alitzer et al. 2003).

Some socially transmitted diseases may be host-density-independent if transmission is a function of the frequency of interactions between host individuals.

Frequency-dependent transmission is likely if infection occurs when hosts seek each other out, either for mating or social aggregations (Anderson & May 1991, Nunn et al.

2008, McCallum et al. 2009) or if the pathogen can survive in an environmental reservoir

(Briggs et al. 2010). Frequency-dependent pathogens are particularly insidious and can drive their host populations to extinction because transmission still occurs at low host- population densities (Getz & Pickering 1983, Lockhart et al. 1996). Social interactions, therefore, reflect an evolutionary trade-off that can fluctuate depending on environmental conditions and pathogen prevalence within the host population (Silk 2007, Mitchell, et al.

2008).

Bats (Chiroptera) have potential as model organisms for studies of pathogen transmission among highly social, colonial hosts, and for understanding the role social behaviour and thermoregulation have on host-pathogen dynamics (Webber et al. 2016,

2017). Bats are heterothermic endotherms (Fenton & Barclay 1980) and exhibit an enormous range of group sizes and social systems ranging from completely solitary to colonies of millions of individuals (Webber et al. 2017). Understanding host-pathogen dynamics of bats has become especially urgent with the emergence of white-nose syndrome (WNS). Populations of hibernating bats in Canada and the United States are currently threatened by WNS, which is caused by the invasive fungal pathogen,

Pseudogymnoascus destructans (Blehert et al. 2008, Frick et al. 2010). WNS is an

12 infectious skin disease that causes bats to rewarm from torpor too frequently during hibernation, and expend extra energy during winter torpor bouts, drastically depleting winter fat stores and resulting in mass mortalities at hibernation sites (Reeder et al. 2012,

Warnecke et al. 2012, McGuire et al. 2017). Bats contract WNS through direct contact with infected individuals or substrates within hibernacula (Lorch et al. 2011). Since its introduction from Eurasia¸ where it is an endemic pathogen of multiple bat species

(Wibbelt et al. 2010, Puechmaille et al. 2011b), P. destructans has caused tremendous declines of naïve populations of North American hosts (Frick et al. 2010, Frick et al.

2015). For example, Frick et al. (2015) found that, in some hibernacula, the disease has caused local extirpation, with mortality rates of 91%, on average, for little brown bats

(Myotis lucifugus). As of January 2013, it was estimated that 5.7 to 6.7 million bats have been killed by WNS (U.S. Fish & Wildlife Service 2013) and this number almost certainly dramatically underestimates the impacts of WNS to date.

Little brown bats are one of the most studied and, until the emergence of WNS, were likely the most abundant bat species in North America (Harvey et al. 2011). During the months of May and June little brown bats form maternity colonies of up to hundreds of reproductive females and their pups, along with a few non-reproductive individuals.

Little is known about male behaviour during the maternity season, but they are presumed to be mostly solitary throughout their range (Harvey et al. 2011). During the late summer and early fall, males and females congregate outside of hibernation sites, likely to mate and introduce juveniles to potential hibernacula (Hall & Brenner 1968, Agosta et al.

2005). Little brown bats hibernate in caves and mines during winter and are one of the species hardest hit by WNS (Harvey et al. 2011). Little brown bats, along with northern-

13 long eared (Myotis septentrionalis) and tricolored bats (Perimyotis subflavus), were recently listed as “endangered” by the Committee on the Statues of Endangered Wildlife in Canada under Schedule 1 of the Species at Risk Act in Canada (Government of Canada

2016).

To reduce the impacts of WNS, numerous chemical and biological management solutions have been designed and tested (Chaturvedi et al. 2011, Puechmaille et al.

2011a, Robbins et al. 2011, Raudabaugh & Miller 2013, Cornelison et al. 2014, Hoyt et al. 2015, O’Donoghue et al. 2015, Pannkuk et al. 2015, Dorville et al. In prep.) but no effective treatment protocols are currently available. At the habitat level, several mitigation techniques have been proposed including: culling of infected individuals

(Langwig et al. 2015), creating warm micro-habitats within caves where bats can save on energetic costs during periodic arousals (Boyles & Willis 2010), and inhibiting P. destructans growth within caves by altering the microclimate (e.g. temperatures, humidity, Shelley et al. 2013, Grieneisen et al. 2015) or applying UV lighting (Palmer et al. 2018). However, these approaches would require continued interventions that may not be sustainable for the long-term (Kilpatrick 2006). Evolutionary rescue may provide a more viable solution for recovery of bat populations affected by WNS (Maslo &

Feffernan 2015). Bat populations near the epicentre of WNS have begun stabilizing following their initial population crashes, albeit at dramatically reduced population sizes

(Dobony et al. 2011, Langwig et al. 2012, 2017). This stabilization suggests the possibility of the evolution of resistance to (i.e., remaining uninfected by a pathogen), or tolerance to (i.e., harbouring the pathogen but limiting its impacts) the pathogen

(Langwig et al. 2017). Survivors of WNS that are resistant or tolerant may pass these trait

14 on to their offspring increasing the frequency of these traits within populations (Maslo &

Feffernan 2015, Carlson et al. 2016). If evolutionary rescue is a promising avenue for recovery of bat populations affected by WNS (Maslo & Feffernan 2015), then encouraging survival and reproduction of WNS survivors should be a critical priority for management (Donaldson et al. 2017).

Habitat enhancement can be a valuable conservation tool for threatened species, including those impacted by the effects of disease (Knaepkens et al. 2004, Savard &

Robers 2007, Kapust et al. 2012, Wilcox & Willis 2016, Tripp et al. 2017). For example, prairie dogs (Cynomys ludovicianus) susceptible to plague (caused by the Yersinia pestis bacterium) benefited from reduced disease impacts after their burrows were treated with insecticide to kill fleas, the vector of the pathogen (Tripp et al. 2017). Habitat enhancement also improved the reproductive success of endangered European bullhead

(Cottus gobio) that are experiencing population declines caused by habitat degradation

(Knaepkens et al. 2004). For bats, enhancement of summer roosting habitat could be a valuable conservation tool if high quality roosting habitat can reduce thermoregulatory energy expenditure and/or torpor expression and increase healing rates and offspring growth rates. For species that regularly use anthropogenic structures (e.g., little brown bats), warm microclimates could be provided by insulating or painting roosts to retain solar heat or even by installing heating panels (Wilcox & Willis 2016). For species more dependent on natural roosts like tree hollows (e.g. Myotis septentrionalis), implementing habitat enhancement would be more challenging but evidence supporting this strategy would justify more effort to understand forest roost requirements of these species.

Habitat enhancement could be an effective management tool for WNS but could also be detrimental if it increases the potential for transmission of P. destructans or other naturally occurring pathogens and parasites of WNS-affected bats. Webber et al. (2016) showed that collapsing large, relatively fluid social networks of bats spread among many natural roosts, into a smaller number of more stable, large colonies can increase the risk of pathogen transmission and proliferation within bat colonies. Enhanced, relatively warm summer roosting habitat could also reduce torpor expression and, therefore, increase overall activity of individuals resulting in more opportunities for bats to contract pathogens and parasites from the environment or each other. Thus, while roosting habitat enhancement has potential to help bats recover from WNS (Maslo & Feffernan 2015,

Wilcox & Willis 2015), it could be counterproductive if it increases the risk of transmission of P. destructans or other pathogens and parasites.

Objectives and Hypotheses

The objective of my thesis was to test the broad hypothesis that habitat quality, via its effects on torpor expression and aggregation, can influence transmission of wildlife pathogens. I also aimed to shed light on the possibility that enhancement of summer roosting habitat could reduce energetic costs of thermoregulation and, therefore, help survivors of WNS.

For my first data chapter (Chapter 2) I tested the hypothesis that high-quality (i.e., relatively warm), roosting habitat reduces reliance on torpor for individual bats, which, in turn, increases activity and the risk of pathogen acquisition from an infected substrate. I predicted that bats provided with high quality, warm roost boxes would: 1) reduce their

16 use of torpor during the active period at night, and spend more time actively investigating potential roosts; and 2) after an exposure period, exhibit higher intensity of infection with a proxy pathogen that I applied to substrates within the testing environment.

For my second data chapter (Chapter 3) I tested the hypothesis that limited high- quality roosting habitat increases passive aggregation for groups of bats and reduces their use of torpor which, in turn, increases the risk of pathogen acquisition. I predicted that bats provided with a relatively rare warm roost would: 1) form larger aggregations in the warm roost; 2) use less torpor; and 3) have a greater intensity of infection with a proxy pathogen that I applied to one randomly selected individual within their social group.

For both experiments I housed bats in outdoor flight enclosures and experimentally manipulated habitat quality by heating a subset of roost boxes in the enclosures. I then quantified the influence of roost temperature on expression of torpor and, for Chapter 3, aggregation size. I used non-toxic, ultraviolet (UV) fluorescent powder as a harmless proxy for a contact pathogen to assess the influence of torpor use and passive aggregation on pathogen transmission from substrates and among individuals.

Habitat quality may influence the transmission of wildlife pathogens via its effects on habitat selection, passive aggregation, and torpor expression. The findings from my study suggest that artificially heating roosts, along with protecting natural roosts with warm microclimates, could improve recovery rates and juvenile development of bats recovering from WNS. Understanding the impacts of environmental variation on the transmission of pathogens to their host species has implications for the design of conservation strategies and could enable managers to both target habitat necessary for

17 protection and find areas where habitat could be artificially improved to facilitate recovery of imperiled species.

Ethical statement

All procedures were conducted under Manitoba Conservation Wildlife Scientific

Permit number WB16368 and approved by the University of Winnipeg Animal Care

Committee. Although the nearest confirmed finding of P. destructans was over 200 km from my study site (northern Minnesota, www.whitenosesyndrome.org, 2016) at the time of my study, I still followed decontamination protocols for researchers outlined by the

U.S. Fish and Wildlife to control the spread of P. destructans

(www.whitenosesyndrome.org, 2016).

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CHAPTER 2: EFFECTS OF ROOST TEMPERATURE ON TORPOR

EXPRESSION AND PATHOGEN ACQUISITION IN SOLITARY

LITTLE BROWN BATS

Abstract

Protection of habitat can improve survival and reproductive fitness of threatened and endangered wildlife, particularly if that habitat helps individuals maintain energy balance. Temperate bats are heterothermic and rely on torpor to save energy during winter hibernation and, to a lesser extent, during summer. However, torpor delays parturition and slows lactation for females, and inhibits sperm production for males, so reproductive bats should select warm roosts to help them avoid torpor. Torpor may also slow healing rates, which could have implications for bats that survive the winter with white-nose syndrome (WNS), a devastating fungal skin disease impacting hibernating North

American bats. WNS survivors must emerge from hibernation and initiate reproduction while also healing from extensive wing damage caused by the disease. Warm roosting habitat could help WNS survivors avoid torpor to heal and reproduce more quickly, enhancing population recoveries. However, reduced torpor expression and increased activity and exploration could increase the chance of bats acquiring pathogens and parasites from substrates in their environment. I tested the hypotheses that warm roosting habitat: 1) reduces use of torpor by endangered little brown bats (Myotis lucifugus); but

2) increases the risk of pathogen acquisition from substrates in the environment. I captured bats from a fall swarm and housed individuals in outdoor flight enclosures equipped with either four heated or four unheated roost boxes. I quantified torpor expression using skin temperature dataloggers and used ultraviolet (UV) fluorescent

29 powder as a proxy pathogen which I applied to one of the four roost boxes in each tent.

Bats provided with warm roosts used less torpor (p<0.0001), but the amount of time a bat spent in torpor (p=0.26), had no effect on intensity of infection with the proxy pathogen.

My data highlight roost temperature as a driver of torpor expression in little brown bats but suggest that heated roosts will not speed rates of pathogen or parasite acquisition from the environment. This result supports the potential of enhancement of summer roosting habitat as a management strategy for WNS.

Introduction

Habitat quality can affect survival and reproductive fitness of wildlife (Nagar &

Noordwijk 1992, Bryan & Bryant 1999). Consequently, habitat protection and enhancement have helped conserve declining or endangered species and populations

(Knaepkens et al. 2004, Merz & Setka 2004, Savard & Roberts 2007, Kapust et al. 2012).

Habitat enhancement involves modifying characteristics of a habitat to improve conservation outcomes for an individual species or overall biodiversity (Weller 1989).

Improved population stability and recovery, the goal of habitat enhancement, can be achieved via direct effects of high quality habitat on individual growth rates (Pérez et al.

2008), body size and condition (Pérez et al. 2008, Ardia et al. 2009), and reproductive rates (Knaepkens et al. 2004, Merz & Setka 2004, Savard & Roberts 2007, Kapust et al.

2012). For example, Pérez et al. (2008) found that artificially heating the nest boxes of tree swallows (Tachycineta bicolor) improved body condition and feeding rates of incubating females. Nestlings in heated boxes also had improved body condition compared to those in unheated boxes. Increased nest temperatures may have reduced

30 energetic costs of thermoregulation for females, enabling them to invest more in incubation and provision of offspring (Bryan & Bryant 1999).

Roosting habitat enhancement could be an important conservation strategy for temperate-zone bats. Hibernating bats in North America are experiencing drastic population declines due to an invasive fungal pathogen, Pseudogymnoascus destructans and the resulting disease, white-nose syndrome (WNS) (Frick et al. 2010, Frick et al.

2015). Three species are now listed as endangered in Canada and one as threatened in the

U.S. because of WNS (Government of Canada 2016). P. destructans infects the skin of hibernating bats during the winter causing increased evaporative water loss and metabolic rate during torpor and, ultimately, an increase in the frequency of arousals from torpor during hibernation, resulting in a rapid depletion of winter fat stores and, potentially starvation (Reeder, et al. 2012; Warnecke, et al. 2012, McGuire et al. 2017). The fungus is spread via direct contact with infected individuals or through contact with infected substrates. While P. destructans only grows at relatively cold temperatures characteristic of hibernation, conidia are thought to remain viable and transmissible within and among hibernacula, not only during winter but also during the spring maternity season and late- summer/fall “swarming” season (Ballmann et al. 2017). During fall swarming, before hibernation, bats visit the entrances of hibernacula in large numbers to mate promiscuously and possibly to investigate potential hibernation sites (Hall & Brenner

1968, Agosta et al. 2005). This behaviour results in high contact rates among bats creating potential for transmission of P. destructans (Dobony et al. 2011, Ballmann et al.

2017) and other contact pathogens and parasites (e.g., Webber et al. 2015). Bats also enter and exit hibernacula frequently during swarming, generating additional potential for

31 acquisition of parasites and pathogens like P. destructans from environmental substrates

(Dobony et al. 2011, Langwig et al. 2015, Carpenter et al. 2016, Ballmann et al. 2017).

Heterothermic endotherms (i.e., mammals and birds), including bats, often use torpor to reduce metabolic rate and body temperature (Tb) and save energy when food is scarce or ambient temperatures (Ta) are low (Lyman et al. 1982, Humphries et al. 2003,

Geiser 2004). The primary benefit of torpor is reduction in energy expenditure, but a range of other benefits could enhance survival and reproductive fitness (reviewed by

Geiser & Brigham 2012). Arid-zone mammals and birds use torpor to reduce evaporative water loss and endure droughts. For example, stripe-faced dunnarts (Sminthopsis macroura) reduce evaporative water loss by 23 to 42% by entering torpor (Cooper et al.

2005). During pregnancy, torpor can delay parturition until favourable conditions for lactation (Racey & Swift 1981, Willis et al. 2006). Torpor may also help some heterotherms avoid predation. Stawski and Geiser (2010) found that Australian northern long-eared bats (Nyctophilus bifax) with larger fat reserves used longer and deeper torpor bouts than those with smaller reserves, suggesting that bats in good condition used torpor to reduce foraging requirements and, therefore, risk of predation. This finding suggests that reduced activity enabled by use of torpor could also reduce the risk of encountering and acquiring pathogens and parasites from the environment although, so far, this hypothesis has not been tested experimentally.

Despite its benefits, torpor is also associated with costs. The expression of torpor in free-living animals reflects an optimization balancing these costs and benefits

(Humphries et al. 2003, Jonasson & Willis 2011, Czenze et al. 2017). Torpor slows gestation and inhibits lactation for reproductive females and inhibits sperm production for

32 males, so reproductive individuals tend to avoid torpor compared to non-reproductive conspecifics (Jagiello et al. 1992, Geiser 1996, Lausen & Barclay 2003, Dzal & Brigham

2013). Torpor may slow the last stages of juvenile development so young-of-the-year

(YOY) animals might be expected to avoid torpor if possible. Torpor can also inhibit wound healing and tissue repair (Faure et al. 2009, Cryan et al. 2013). Like reproductive individuals and YOY, heterotherms recovering from injuries, or bats recovering from

WNS in spring, may also benefit from remaining normothermic.

Torpor avoidance appears to influence roost and nest selection decisions of heterothermic endotherms (Holloway & Malcolm 2007, Coombs et al. 2010) presumably to help individuals reduce thermoregulatory energy costs. For instance, rodents select nesting sites like subterranean burrows that buffer against the surface environment

(Begall et al. 2007), and flying squirrels select well-insulated tree cavities (Holloway &

Malcolm 2007). In addition to helping individuals avoid torpor, warm microclimates can also speed growth rates of offspring (Tuttle 1976, McCarty & Winkler 1999). Benefits of warm roosts or nests for torpor avoidance and reproduction suggest that enhancements to roosting or nesting habitat, which reduce normothermic thermoregulatory costs during the reproductive season could be important for the conservation of heterothermic endotherms. This reduction in costs could be especially important for the few bats that survive the winter with WNS. Some WNS-affected individuals survive the disease and emerge from hibernation in spring (Maslo et al. 2015). However, these survivors mount an intense, energetically-expensive and, likely, maladaptive inflammatory response which causes immune-mediated tissue damage (Meteyer et al. 2012). They must, then, spend energy to heal tissues damaged by WNS and this subsequent immune response (Fuller et

33 al. 2011). For females, spring is also normally the time of reproductive investment but if fat reserves must be re-allocated to WNS recovery, this investment could negatively impact reproduction by survivors. Providing bats with relatively warm roosts that help them avoid torpor could facilitate faster healing and help bats reproduce earlier in summer, improving the potential for juveniles to survive their first winter (Wilcox &

Willis 2016). By facilitating reproduction of WNS-survivors, roosting-habitat enhancement could also facilitate so-called ‘evolutionary rescue’, the evolution of WNS- resistance or tolerance traits in endangered bat populations (Willis & Wilcox 2014, Maslo et al. 2015, Maslo & Fefferman 2015).

Consistent with the habitat enhancement hypothesis, Wilcox & Willis (2016) used a captive colony of little brown bats to show that bats emerging from hibernation were significantly more likely to select an artificially heated bat house in their flight enclosure over an unheated control bat house. Moreover, bats recovering from WNS showed a dramatically stronger preference for the heated roost compared to healthy individuals.

This preference suggests the possibility that artificially-heated roosts, as well as natural roosts that provide warm microclimates, could be beneficial for WNS survivors. On the other hand, one potential risk of roosting habitat enhancement could be increased risk of contracting parasites and pathogens, including P. destructans, if warm roosts reduce torpor expression and increase overall activity and exploration of the environment.

Torpor is thought to reduce risk of predation for bats (Stawski & Geiser 2010) and could also reduce the chance of encountering and contracting pathogens and parasites from substrates in the environment.

I tested two hypotheses important for understanding the potential of roosting

34 habitat enhancement as a management tool for WNS. First, I tested whether warm roosting habitat reduces the expression of torpor by bats captured during the fall swarming season. I predicted that bats provided with heated roost boxes would use less torpor during the active period at night than bats provided with un-heated, control roost boxes because of improved potential to balance their energy budget. Second, I tested whether reduced reliance on torpor increases the risk of acquiring contact pathogens or parasites from substrates in their environment, presumably because of increased overall activity. I predicted that bats provided with heated roosts would exhibit higher prevalence and intensity of infection with a proxy pathogen (i.e., ultraviolet (UV) fluorescent powder) applied to substrates they would be likely to explore (i.e., potential roost sites) in their environment. Evidence that enhanced, warm roosting habitat can reduce torpor expression without increasing infection risk would support application of this management technique for imperiled populations and/or species of bats.

Methods

All procedures were conducted under Manitoba Conservation Wildlife Scientific

Permit number WB16368 and approved by the University of Winnipeg Animal Care

Committee. The nearest confirmed detection of P. destructans was >200 km from my study site at the time of my study (northern Minnesota, www.whitenosesyndrome.org

2016) but I still followed U.S. Fish and Wildlife Service decontamination protocols for researchers to control the spread of P. destructans (www.whitenosesyndrome.org, 2016).

I conducted 9 three-night trials between 17 August and 10 September 2016. On the first night of each trial I used a harp trap to capture 1 – 2 bats per night outside of St.

George Bat Cave near Fisher River First Nation, Manitoba, Canada (14U 613884

5700551). Since 2008, bats at this site have been marked with Passive Integrated

Transponders (PIT tags) as part of a mark-recapture study and I used a handheld PIT tag scanner (EID-LID 573: Handheld reader, Electronic Identification Systems Inc.,

Brampton, ON, Canada) to identify recaptured bats and eliminate the chance of re-using bats for multiple trials in my experiment. Bats were then placed in cloth bags and within

2 h of capture were transported 13 km, by car, to a field laboratory near Lake St. George.

Once at the field laboratory, I sexed each bat and assessed reproductive status of females as “lactating” if nipples were bare and/or I could express milk, or “non- reproductive” if the hair had grown back around the nipples. I aged bats as adults (>1 year) or YOY (<1 year) based on the degree of ossification in the metacarpal-phalangeal joints of the fourth digit of the wing (Kunz & Anthony 1982). I measured length of the right forearm (± 0.1 mm) using digital calipers (100-300-8 EZ Cal, iGaging Precision

Instruments, San Clemente, CA, USA) and mass (±0.1 g) using an electronic balance (FP

1000, Fuzion Diablo, Barcelona, Spain).

To quantify torpor expression, I recorded each bat’s skin temperature (Tsk) using small temperature dataloggers (0.5°C resolution, iButtons, DS1922L, Maxim Integrated

Products Inc., Dallas, TX, USA) modified to reduce their mass following Reeder, et al.

(2012). Final miniaturized iButtons weighed ~1g. I programmed iButtons to record Tsk every 5 min and attached iButtons using non-toxic latex-based adhesive (294-Osto-bond,

Montreal, Canada) to the dorsum between the scapulae. I did not trim the fur before attaching iButtons. This process likely increased ambient cooling of dataloggers relative to recording Tsk (see results) but, in my experience, skin attachment makes datalogger

36 removal much more difficult and would have increased stress for the animals. To remove iButtons at the end of each trial, I simply cut the fur between the glue and the skin, leaving a patch of slightly shorter fur. After processing, I placed bats in a 20 by 21 by 24 cm stainless steel mesh cage with ad libitum water and felt cloth to hang on until dusk the next day when their experimental trial began.

Beginning at sunset on the day following capture, I randomly assigned each bat to one of two outdoor flight enclosures (length: 4.3m, width: 4.3m, height: 2.0 m; Model

#076-5460-2, Outbound Screen House, Outbound, Toronto, Canada). Each enclosure was outfitted with four custom-built single-chamber roost boxes (33 cm tall by 30 cm wide by

3 cm deep). Roosts were insulated with 3 cm thick Styrofoam insulation (Model #

547658, CodeBord Extruded Polystyrene Rigid Insulation, 3 cm thick, Foamular, Owens

Corning Commercial Insulation, Toledo, OH, USA). Each roost box was open at the bottom, so bats could enter and exit freely and had a hinged front panel, allowing me to open the roost and remove bats at the end of each trial. The back of each roost was equipped with a layer of felt covered by window screening to provide footholds on which bats could hang. In one of the two enclosures (hereafter the control enclosure), roosts were not heated and roost temperature (Troost) fluctuated with outside Ta. In the other enclosure (the heated enclosure), each roost box was equipped with a terrarium heating pad (Model # PT2035, Heat Wave Desert, Exo Terra, Montreal, Canada) positioned under the roosting felt/mesh. Heating pads were connected to outdoor temperature controllers

(ETC-141000-000: NEMA 4X, Ranco ETC, Delphos, OH, USA) set to 32°C. I recorded

Troost and Ta adjacent to flight enclosures using unmodified iButtons set to record every

10 min. Heated roost boxes maintained significantly warmer Troost (21.23 ± 1.79°C)

37 relative to control roosts (16.57 ± 1.89°C; Welch two sample T-test, t = 4.9, df =12.5, p =

0.0003, Figure 2.1)

Figure 2.1: Boxplot of daily average Troost in both heated and control roost boxes in heated and control flight enclosures. Centre lines represent median values, boxes represent the interquartile range, the upper whisker represents the third quartile plus 1.5 times the interquartile range, and the lower whisker represents the first quartile minus 1.5 times the interquartile range. Open circles represent outliers.

Water and mealworms, (larval Tenebrio molitor), gut-loaded with beta-carotene multivitamins (SRP 00300, Herptivite, CA, USA) and a nutrient supplement (Repashy

Superfoods, Repashy Ventures Inc., CA, USA) were provided ad libitum on a small

(71x51x77cm) folding table positioned in the middle of each enclosure.

Prior to introducing bats to either flight enclosure, I randomly selected one roost box in each enclosure for infection with my proxy contact pathogen, UV fluorescent powder (AX-18N, Signal Green, Day-Glo Color Corp. Cleveland, Ohio, USA). Webber

(2016) found that transmission dynamics of this UV powder in this experimental setup were similar to those for real-world contact pathogens or parasites with negative binomial distributions for intensity of infection among hosts. Using a small paintbrush, I applied

100ml of UV powder to the mesh on the back panel of the selected “infected” roost. I applied the powder in a horizontal line, 2 cm wide, 30 cm long and 20 centimetres from the top of the roost. Thus, if a bat thoroughly investigated and/or chose to roost in the infected roost it would have crossed this infected zone and, in turn, become “infected” with the pathogen.

After I applied UV powder to the roosts, at dusk (range: 20:39 – 19:55) on the night of each experimental trial, I weighed each bat, hand-fed them both 10 mealworms and then released one individual into the control enclosure and the second bat into the heated enclosure. After 24 hours, I re-entered the flight enclosures, measured Ta and recorded the roost box in which each bat was found. I never observed bats outside of roost boxes during the day and, for my analyses, I assumed that bats roosted the entire day inside the roost in which I found them upon recapture. After removing each bat from the flight enclosure, I photographed the dorsal and ventral sides of its wing and tail

40 membranes to quantify the prevalence and intensity of infection with the proxy pathogen.

I took photographs under a UV lamp (HQRP, Harrison, NJ, USA) with a digital camera

(Digital Rebel XTi, Canon Inc., Oita, Japan) mounted on a tripod. I included a scaling item (i.e., U.S. penny, diameter = 19.05mm) in each photo to standardize image sizes.

After photographing each bat, I weighed it and injected a uniquely coded PIT tag under the skin between the scapulae to identify recaptured individuals. I then released each bat at the capture site.

Methods for defining when endotherms are “in” or “out” of torpor have been a topic of debate among eco-physiologists (Barclay et al. 2001, Willis & Brigham 2003,

Boyles et al. 2011, Brigham et al. 2011). One approach for comparing torpor among individuals is to calculate so-called degree-minutes of torpor expression by quantifying the area under a Tb or Tsk time-course below some torpor threshold (e.g., active temperature) (Barclay et al. 2001, Lausen & Barclay 2003). This approach has been criticized because it fails to account for Q10 effects (the rate of change in physiological reactions when Tb increases 10℃) which result in larger energetic savings from initial, shallow reductions of Tb compared to smaller energetic savings accrued from reductions of the same magnitude at lower Tb (Willis & Brigham 2003, Brigham et al. 2011).

However, for my study, defining the beginning and end of torpor bouts, or estimating energetic savings, was less important than quantifying overall differences in Tb among individuals and between treatments. Therefore, I used an area-under-the-curve approach, modified from Lausen and Barclay’s (2003) degree-minute calculation. I first interpolated

Tsk values for each 5 min interval between measurements by multiplying each measured

Tsk value by five. I then summed all measured and interpolated Tsk values for each 24-

41 hour trial. In contrast to many studies which depend on some torpor threshold, (e.g.,

Barclay et al. 2001, Lausen & Barclay 2003) this calculation does not directly estimate expression of torpor but, instead, provides a relative index of the defense of high Tsk or, what I term, a normothermy index (NI). In other words, values of NI were higher for bats that spent less time in torpor and more time normothermic.

I used ImageJ (ImageJ v. 1.8.0_66, U.S. National Institutes of Health, Bethesda,

Maryland, USA) to quantify the intensity of infection with UV powder in photographs of bats’ wings. I first standardized images based on the scaling item in each photo, then outlined each wing to quantify total wing area (Fuller et al. 2011). I then summed the area covered with particles of UV powder and divided by total wing area. To facilitate analysis, for one bat that had no detectable UV powder on its wings, I replaced its value of zero with the lowest intensity value I determined for all other bats divided by 2, assuming that this lowest value represented my minimum limit of detection (Croghan &

Egehy 2003). Intensity of infection fit a negative binomial distribution, so I used a logit transformation to achieve normality (see results). I then assessed the effect of degree minutes on infection intensity (Webber 2016).

I used R (version 3.2.3, GUI 1.51, R Development Core Team 2015) for all statistical analysis. To test whether bats in the heated enclosure were less likely to use torpor, I used a linear mixed effects model with NI as a response variable, daily average

Troost as a fixed effect, and trial number as a random effect to account for potential pseudo-replication of data across trial days. Neither sex nor mass was included in my models. Sex and body mass could affect torpor expression (Chapter 3) but, because of my sample size (n=15), I would not have had the statistical power to detect effects of sex or

42 mass. To test whether torpor expression influenced pathogen acquisition, I used a linear mixed effects model with intensity of infection (i.e., logit-transformed proportion of wing covered with UV powder) as the response variable, NI as a fixed effect and, again, trial day as a random effect. I examined residual plots to ensure all models met standard assumptions. I assessed significance at the α=0.05 level and all values are reported as the mean ± SD unless otherwise specified.

Results

Ambient temperature ranged from 10.0°C to 35.5°C throughout the study with average daily maxima of 24.1±6.2°C and minima of 13.8±1.8°C. Sunrise times ranged from 05:26 to 06:01, and sunset times from 19:03 and 19:51 (National Research Council

Canada 2016). I ran trials on 15 individuals (n = 9 female, 6 male, Table 2.1). My Tsk measurements underestimated normothermic Tb because I did not trim the fur underneath dataloggers, but I was still able to record clear torpor/arousal cycles (Figure 2.2). Tfur was typically relatively low during the active phase at night but often increased rapidly when bats entered heated roosts in the morning (Figure 2.2).

2 I found a significant positive effect of average Troost on NI (r =0.81, Table 2.2,

Figure 2.3). Based on calculated parameter estimates, the model predicted a 603 degree- minute increase in NI for each 1°C increase in daily average Troost. The NI incorporates both depth and duration of torpor so that a 603 degree-minute difference could represent a range of possible responses (e.g., an increase of Tb by 10°C for 60 minutes, 2°C for 300 minutes, etc.). However, the ultimate result was that higher values of Troost were associated with higher values of Tsk.

Mass Intensity of Average Average Trial Treatment Sex NI (g) Infection Troost Ta

1 Heated Female 8.9 0.0061 23.9 20.7 44097.26 1 Control Female 9.4 0.0002* 20.3 20.7 41915.47 2 Heated Female 9.7 0.0946 21.4 17 44057.62 2 Control Male 8.5 0.0041 16.6 17 39277.00 3 Heated Female 9.4 0.0132 19.6 15.1 42191.63 3 Control Male 11.3 0.0008 14.4 15.1 39859.21 4 Control Female 8.5 0.0003 17.2 17.8 40240.57 5 Heated Female 9 0.0119 23.8 18.8 44164.20 6 Heated Female 8.4 0.0006 21.1 16 43787.62 6 Control Male 8.6 0.0304 15.2 16 39768.35 7 Heated Male 8.7 0.0232 21.1 17 43029.67 7 Control Female 8 0.0026 16 17 39505.16 8 Heated Male 12.1 0.0028 19.7 15.8 43214.98 8 Control Male 12.2 0.0007 16.2 15.8 39467.08 9 Heated Female 11.8 0.0036 19.3 13.5 43975.31

Table 2.2: Summary of linear mixed effects model assessing effects of average Troost

(with trial as a random effect, p<0.0001) on normothermy index of 15 little brown bats

(n=6 male, 9 female) over 9 24-hour trials.

Degrees of Variable Coefficient Standard Error t-value p-value Freedom Average 603 78.9 7.64 5 <0.0001 Troost

21:00 7:00 17:00 21:00 7:00 17:00

Intensity of infection fit a negative binomial distribution (Figure 2.4A) but I achieved normality using a logit transformation (Shapiro-Wilk normality test, W= 0.97, p=0.92, Figure 2B). Intensity of infection averaged 1.50 ± 2.43% wing coverage and ranged from 9.46% to 0.03% coverage with one individual exhibiting no infection. I found no effect of individuals’ NI values on logit-transformed intensity of infection

(r2=0.10, Table 2.3, Figure 2.5).

Degrees of Variable Coefficient Standard Error t-value p-value Freedom NI 0.0003 0.0002 1.33 5 0.24

A B

Figure 2.4: Histograms of (A) un-transformed intensity of infection (i.e., proportion of the wing covered with UV powder), and (B) logit-transformed intensity of infection for

15 little brown bats during environmental-substrate transmission infection experiment.

Discussion

I found support for my first hypothesis that Troost influences torpor expression by little brown bats. Bats provided with warmer roosts maintained higher values of Tsk indicative of reduced torpor expression. Despite a pronounced effect of Troost on use of torpor, however, I found no support for my second hypothesis that a reduction in torpor use increases the risk of acquiring contact pathogens or parasites from substrates in the environment. These results lend support to the potential of roosting habitat enhancement as a management tool for imperiled or endangered populations of temperate-zone bats.

White-nose syndrome causes increased torpid energy expenditure and frequent arousals in hibernating bats, leading to premature depletion of fat stores, and presumably starvation (Warnecke et al. 2012, Verant et al. 2014). Bats that survive the winter emerge from hibernation with severely reduced energy reserves and must begin the energetically- expensive task of healing while food is still scarce, and Ta is still cold (Wilcox & Willis

2016). Warm Tb is known to accelerate wound healing in heterotherms (e.g. Anderson &

Roberts 1975, Faure et al. 2009) and bats healing from skin damage caused by WNS may select warm roost microclimates to increase healing rates while reducing energetic costs of thermoregulation (Wilcox & Willis 2016). Wilcox and Willis (2016) gave bats recovering from WNS access to both heated and unheated roost boxes, and the infected bats virtually always selected the artificially heated roost. Although bats in my study were all negative for WNS, my findings build on those of Wilcox and Willis (2016) by demonstrating that bats provided with relatively warm roosts reduce torpor expression which could be beneficial for individuals recovering from WNS. If bats can defend higher Tb at reduced energetic costs, this strategy could help them allocate more energy to

52 healing and/or reproduction. This benefit, in turn, could be important for helping foster evolutionary rescue of populations impacted by WNS. Bats that survive the winter with the disease may exhibit heritable behavioural and/or physiological traits that favoured their survival. Offspring born to those potentially resistant or tolerant individuals could inherit those favourable traits and help foster an evolutionary response to the disease

(Gonzales et al. 2013, Maslo & Fefferman 2015, Maslo et al. 2015, Wilcox & Willis

2016). This result suggests that protecting existing summer habitats that provide warm roost microclimates, and potentially enhancing roosting habitats via artificially heating or other means to increase Troost, could help support evolutionary rescue and population recovery.

One potential downside of roosting habitat enhancement is the potential to increase activity and exploration of the environment by bats and, therefore increase risk of encountering and contact pathogens and parasites from environmental reservoirs. Like

Webber (2016), I found that transmission dynamics of UV fluorescent powder introduced to roosting substrates in a flight enclosure reliably mimicked patterns of acquisition for naturally occurring pathogens and parasites. Intensity of infection typically shows a negative binomial distribution for many pathogens and parasites (e.g., Shaw et al. 1998) and this pattern matches what Webber (2016) and I observed for UV powder. Despite this predicted pattern of infection, however, I found no evidence that warm roosts and/or reduced torpor expression, increased risk of infection for individuals. Although more work is needed, the fact that bats, especially individuals recovering from WNS, prefer artificially heated roosts (Wilcox & Willis 2016), that bats with heated roosts maintain higher Tb and express less torpor, and that reduced torpor expression does not appear to

53 increase risk of pathogen acquisition from environmental substrates, lend support to the habitat enhancement hypothesis that warm roosting microclimates could help bat populations recover from WNS and other threats.

Although my results support habitat enhancement as a management tool, there are limitations to my experiment that should be addressed before this management approach is implemented on a wide scale. For one, my study took place over one-month during the late-summer, early-fall swarming period so results may not apply to other times of year

(e.g., spring WNS recovery and reproduction). Although large enough to detect large effects of heated roosts on torpor expression, my sample size was relatively small because there was only a very limited time window during the fall swarm period, during which I could conduct my experiment. This could have limited my ability to detect effects of torpor on pathogen transmission. According to my power analysis, my sample size would have had to be larger (37 bats, instead of my 15) in order to detect even a large effect of NI on pathogen acquisition. Based on an a priori power analysis, I recommend that future studies aim for sample sizes of 40-50 individuals in order to detect large effects, biologically meaningful effects if they exist. The timescale of each trial was also relatively short. Little brown bats do not survive well in captivity for long periods during the active season so to avoid impacts on this endangered species, I limited the duration of my experimental trials. It is possible that, over a longer time period, I would have observed an effect of Troost and torpor expression on risk of pathogen acquisition.

The spatial scale of my trials was also relatively small compared to the very large home ranges of little brown bats in my study region (Habrich et al. in prep.). This small scale could have led to unusual behaviour by bats in my study that reduced potential effects of

54 torpor expression on pathogen acquisition. On the other hand, enclosure size could have helped counteract potential effects of short-duration trials. Although my trials were brief, bats were housed in a relatively small space, increasing the chance that they would have encountered the infected substrate in their enclosure. Another factor which clearly differed in my experiment compared to the wild was provision of ad libitum food and water for all bats in my experiment. For free-ranging bats, warm roosts during periods of cold Ta may help reduce thermoregulatory costs of remaining normothermic but if very little food and only warm roosts are available, the inability to enter torpor could prevent bats from balancing their energy budgets. Support for the habitat enhancement hypothesis from this study and others (e.g., Wilcox & Willis 2016) suggest that similar experiments should be attempted on larger spatial and longer temporal scales with free-ranging bats.

Artificial heating of roost boxes is one habitat enhancement option for some bat species (e.g., little brown bats) which regularly use anthropogenic structures. However, other endangered bat species rarely use these kinds of structures (e.g., northern long- eared bats, M. septentrionalis). For these species, identifying, protecting and enhancing habitat features which naturally increase Troost could be useful in terms of management. A meta-analysis conducted by Kalcounis-Ruppell et al. (2005) found that bats roosted in tall trees with greater diameter at breast height in stands with open canopy and high snag density. Features such as southern facing slopes and mixed age stands with abundant emergent trees already excavated may also provide warm microclimates. BrandenbarkTM

(Copperhead Environmental Consulting, Paint Lick, KY, USA), a new form of artificial roost made from polyurethane elastomeric Flex-Bark (Replications Unlimited,

Hazelwood, MO, USA), may provide a more stable microclimate than natural roosts.

Brandenbark roosts have already been shown to buffer against cool Ta and their use has been confirmed for northern-long eared bats and Indiana bats (M. sodalis; Adams et al.

2015). The preservation and enhancement of high quality natural summer roosting habitat, and the creation of artificially enhanced habitat, could potentially be important management approaches for protection and conservation of bat species at risk.

I found that Troost affects torpor expression of little brown bats. Bats with access to warmer roosts used less torpor than bats with only unheated control roosts. By using artificially warm roosts, bats presumably used less energy maintaining normothermia and could potentially invest more energy in healing from injury or disease and/or reproduction. This finding could, in turn help WNS-impacted populations recover and evolve resistance to, or tolerance of, WNS. While bats in warmer roosts used torpor less, they were not at greater risk of acquiring a contact pathogen from substrates in their environment compared to bats without access to warm roosts. Taken together, these results suggest that artificially heating bat houses and identifying, protecting and enhancing features of natural roosts which increase Troost, could be an effective management approach for bat populations impacted by WNS and other threats.

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CHAPTER 3: EFFECTS OF ROOST TEMPERATURE ON

AGGREGATION AND TORPOR EXPRESSION BY GROUPS OF LITTLE

BROWN BATS

Abstract

Habitat heterogeneity can lead to variation in aggregation patterns of conspecifics, which can result in costs or benefits for individuals. For endothermic animals, aggregation may increase access to high quality habitat or reduce energetic costs of thermoregulation but could also increase risk of pathogen or parasite acquisition. During the active season, female bats aggregate in warm roosts, huddle with conspecifics, and/or enter torpor (i.e., a state of reduced body temperature (Tb) and metabolism) to reduce thermoregulatory costs. Artificial roosts, such as bat houses, are often deployed as a management tool for bat populations. However, if bat houses increase aggregation beyond normal levels, they could increase pathogen or parasite transmission which could be especially problematic for species affected by white-nose syndrome (WNS). I used little brown bats (Myotis lucifugus) to test three hypotheses about effects of roost quality on aggregation, torpor expression and pathogen transmission: 1) limited, high quality, warm roosting habitat increases bat aggregation and 2) decreases torpor use; and 3) reduced torpor expression and increased aggregation increases pathogen transmission. I predicted that bats provided with access to a high quality, warm roost box would form larger groups, express shorter, shallower torpor bouts, and exhibit greater intensities of infection when a proxy pathogen was introduced to the group. To test these predictions, I housed groups of 10 bats in an outdoor flight enclosure and provided heated or unheated

(control) roosts. I quantified roost occupation each day, assessed torpor expression using

64 temperature data-loggers attached to the skin, and used ultraviolet (UV) fluorescent powder as a proxy for a contact pathogen. Bats disproportionately aggregated in heated roosts (p<0.0001) and, after controlling for trial number, I found a significant effect of an interaction between roost temperature (Troost) and aggregation (p<0.0001) on torpor use.

Troost had a stronger effect on torpor use (p=0.0001) for small groups than for medium

(p=0.81) or large groups (p=0.020). However, pathogen transmission was not affected by either group size (p=0.80) or torpor expression (p=0.68). My findings suggest that roost boxes, artificially heated structures, and other warm microhabitats could help reduce energetic costs for bats affected by threats like WNS, without increasing transmission of contact pathogens or parasites. More work is needed to understand these relationships for larger group sizes over longer time scales.

Introduction

Animals tend to aggregate in areas of high quality habitat (Weir & Davison 1966,

Johnson et al. 2000, Begall et al. 2007) which provide access to resources, protection from predators and, for endotherms, roost or nest sites that buffer against fluctuations in ambient temperature (Ta). For example, Eurasian badgers (Meles meles), a typically solitary species, will aggregate in areas with high food abundance when food distribution is patchy (Johnson et al. 2000), and the distribution of watering holes during dry periods influences the presence and abundance of many African mammals (Weir & Davison

1966). Small-bodied endotherms often select nest or den sites that minimize exposure to low Ta to decrease thermoregulatory costs, like subterranean burrows (Begall et al. 2007), or well-insulated tree cavities (Coombs et al. 2010).

Communally-living or colonial animals may also directly benefit from cooperative interactions with other conspecifics. Benefits can include increased overall reproductive success (Malcolm & Marten 1982, Burrows 1995, Roulin & Heeb 1999,

Russell et al. 2002, Courchamp & MacDonald 2003, McGowan et al. 2003), improved foraging efficiency (Packer & Ruttan 1988, Wilkinson & Boughman 1998), information transfer about foraging habitat (Wilkinson 1992) or other nesting/roosting habitat

(Chaverri et al. 2010), and reciprocal food sharing (Wilkinson 1985). The presence of multiple individuals in a den or roost can decrease thermoregulatory costs and water loss of individuals by raising Ta in the site by reducing the surface area of individuals exposed to ambient air or cold substrates (Arnold 1990, Hwang et al. 2006, Willis & Brigham

2007, Boyles et al. 2008, Boratynski et al. 2015). Social thermoregulation, or mutual warming, occurs when endotherms communally nest to reduce thermoenergetic costs associated with maintaining body temperatures (Tb) (Madison 1984, West & Dublin

1984, Koprowski 1998).

Many endotherms exhibit facultative heterothermy and use short bouts of torpor, periods of reduced Tb and metabolism, to save energy during times of low food or water availability and/or low Ta. For example, desert hedgehogs (Paraechinus aethiopicus) in

Saudi Arabia frequently enter torpor bouts during spring and winter, occasionally for over

24 hours, and allow Tb to fall within 2-3°C of Ta. In this species, torpor likely reflects a response to the arid environment or the unpredictability of food resources (Boyles et al.

2017). Australian northern long-eared bats (Nyctophilus bifax) are also known to use daily torpor, even during periods of high food abundance and high Ta. Stawski and Geiser

(2009) hypothesized that, because N. bifax roost in cryptic locations that are likely safe

66 from predators, the use of torpor allowed bats to reduce activity and the amount of time spent foraging, which could ultimately reduce their risk of predation. Thus, in addition to energy savings, the ability to express torpor may have allowed bats in good condition to reduce their risk from predation (Stawksi and Geiser 2009, Geiser and Brigham 2012).

Torpor can result in energy savings and a range of other benefits (Geiser &

Brigham 2012), but it can also be detrimental. Humphries et al. (2003) showed that artificially supplementing eastern chipmunks (Tamias striatus) caused an increase in time spent normothermic during winter hibernation. Individuals supplemented with additional food were normothermic more than twice as frequently and defended higher temperatures

(5°-10°C) during torpor bouts compared to non-supplemented conspecifics. These results show torpor may be avoided when it is not energetically necessary (e.g. periods of high food abundance), and that even during hibernation, animals may benefit from defending warmer Tb when possible. Costs of torpor for reproductive individuals are also well known. Pregnant and lactating mammals avoid torpor compared to non-reproductive conspecifics (Geiser 1996, Lausen & Barclay 2003, Dzal & Brigham 2013) because torpor slows fetal development (Hoying & Kunz 1998) and milk production (Wilde et al.

1999). Torpor may also slow the final stages of juvenile growth after independence, interrupt sperm development for males (Jagiello et al. 1992) and slow healing rates from injury (Cryan et al. 2013). Therefore, during some circumstances, individuals other than reproductive females may also benefit from avoidance of torpor expression.

During winter most temperate-zone bats use extended bouts of torpor, or hibernation, for weeks to months (Fenton & Barclay 1980, Jonasson & Willis 2012,

Norquay & Willis 2014, Czenze et al. 2017). Hibernating bats in North America face

67 many threats including habitat loss (Kunz & Lumsden 2003), climate change (Humphries et al. 2002), industrial wind energy (Arnett et al. 2008), eviction from building colonies, and, most urgently, infectious disease (Frick et al. 2010, Langwig et al. 2012, Warnecke et al. 2012). North American bats are experiencing drastic population declines from the invasive fungal pathogen, Pseudogymnoascus destructans, which causes white-nose syndrome (WNS), an infectious skin disease. Bats with WNS rewarm from torpor too frequently during hibernation and deplete their winter fats stores prematurely, resulting in mass mortalities at hibernation sites (Reeder et al. 2012, Warnecke et al. 2012). The fungus is spread from infected individuals and substrates to uninfected individuals via direct contact (Lorch et al. 2011), and transmission may be affected by the degree of aggregation by hosts (Wilder et al. 2011, Langwig et al. 2012).

Most proposed management approaches for WNS involve actions that would be implemented in winter to reduce fungal loads on bats or substrates in hibernacula, and/or reduce disease severity and improve winter survival of bats (e.g., Boyles & Willis 2010,

Cheng et al. 2016, Cornelison et al. 2014, Palmer et al. 2018). However, any such intervention would be extremely challenging from a logistic perspective and so-called

‘evolutionary rescue’ may be the best hope for recovery of afflicted populations (Maslo &

Feffernan 2015). Some colonies near the original, 2007 outbreak of the disease in the northeastern U.S. are beginning to stabilize at 5-30% of their original population size

(Dobony et al. 2011, Langwig et al. 2012, Frick et al. 2015). This stabilization suggests the possibility that surviving individuals possess heritable traits and genotypes that, in the face of an intense selective sweep, have increased in frequency within remnant populations (Willis & Wilcox 2014, Langwig et al. 2017). If WNS survivors can pass on

68 adaptive traits to their offspring, this could result in evolutionary rescue of populations

(Maslo & Feffernan 2015, Carlson et al. 2016, Donaldson et al. 2017). Survivors of WNS emerge from hibernation in the spring but face the energetically expensive process of healing and reproduction. Roosting habitat that helps these survivors reduce torpor expression, may help speed recovery (Faure et al. 2009, Cryan et al. 2013) and increase reproductive success of survivors (Bryan & Bryant 1999) which could enhance the potential of evolutionary rescue and population recoveries. Protection and enhancement of spring and summer habitats could be just as, if not more important for population recoveries than treatments or other winter management actions (Wilcox & Willis 2016).

Some bat species impacted by WNS will regularly use anthropogenic structures and artificial habitats, like bat boxes (Kunz 1982). Wilcox and Willis (2016) used one of these species, the little brown bat (Myotis lucifugus), to test the habitat enhancement hypothesis that active-season roosting habitat could be important for helping bats recover from WNS. They provided a heated roost box, warmed to near-thermoneutral roost temperature (Troost), and a control roost box without heating (Troost = 18°C) to each of two groups of bats (i.e., recovering from WNS versus control) in spring, immediately following hibernation. Both groups preferentially chose the heated bat box in their respective enclosures, but the WNS group was dramatically more likely to use the heated roost box compared to control bats. Wilcox and Willis (2016) used an energetic model to show that roosts maintaining thermoneutral Troost most or all of the time would allow an average bat, defending normothermia, to reduce daily energy expenditure by 75 – 81% under typical spring conditions in central Canada. If enhanced roosting habitat reduces the energy required to defend high Tb and the expression of torpor, this approach could

69 help bats recover from WNS in spring, reproduce earlier summer, and better prepare for hibernation in fall. However, to date no study has quantified torpor expression of bats provided with artificially enhanced roosting habitat.

One potential risk of using enhanced summer roosting habitat is disruption of social networks and increased aggregations of bats beyond natural colony sizes.

Increasing social group size or increasing connections among more individuals in a social network, can increase pathogen or parasite transmission for bats and other animals (Ryder et al. 2007, Nunn et al. 2015, Webber et al. 2017). Many bat species exhibit fission/fusion social dynamics, with a large social group splitting into multiple subgroups on a day-to-day basis, often in multiple roost structures within the same local area (Kerth

& König 1999, Willis and Brigham 2004, Kerth et al. 2011). However, as bats switch roosts over time, individuals from each sub-group split apart and fuse with other individuals, maintaining cohesion within a large colony despite roosting in relatively small sub-groups. This behaviour is thought to help individual bats maintain a wide range of potential roosting partners for social thermoregulation while reducing overall loads of pathogens and parasites within the colony (Kerth & König 1999, Willis & Brigham 2004,

Kerth 2008). Webber et al. (2016) used epidemiological models based on social network data from colonies of big brown bats (Eptesicus fuscus) to show that collapsing a dispersed fission/fusion network of small subgroups into a smaller number of roosting aggregations with more individuals in the same roost structure, increased the potential for pathogen transmission and loads within the overall colony. If roosting habitat enhancement causes a similar pattern of increased aggregation for bats recovering from

WNS, it could potentially increase transmission of P. destructans and/or other parasites

70 and pathogens which could, in turn, be counter-productive for conservation and management.

I used an enclosure experiment with little brown bats to test three related hypotheses about potential costs and benefits of artificial habitat as a management tool for bats. First, I tested whether high quality, warm roosting habitat increases passive aggregation by groups of bats. I predicted that, if groups of bats were provided with limited, artificially heated roosting habitat, the sizes of their aggregations would increase in these warm roosts. Second, I tested whether limited, warm roosting habitat, and/or greater aggregation sizes, influenced thermoregulation. I predicted that bats roosting in warmer roosts and/or larger groups would use torpor less than those in cold roosts and small groups. Finally, I tested whether aggregation and/or torpor expression would increase risk of pathogen acquisition. I predicted that bats roosting in larger aggregations, and using less torpor, would exhibit increased intensity and prevalence of infection following introduction of a proxy pathogen (i.e., ultraviolet (UV) fluorescent powder) via one infected individual within the group.

Methods

All procedures were conducted under a Manitoba Conservation Wildlife Scientific

Permit number WB16368 and approved by the University of Winnipeg’s Animal Care

Committee. I followed WNS decontamination protocols outlined by the U.S. Fish and

Wildlife Service (www.whitenosesyndrome.org, 2016) to slow the spread of P. destructans, although at the time of the study, the nearest confirmed case of WNS was

200km from my study site (northern Minnesota, www.whitenosesyndrome.org, 2016).

From 17 August through 10 September 2016 I conducted 11 enclosure trials, each lasting 24 hours. On the first night of each trial, I captured 10 little brown bats outside of a known hibernaculum in central Manitoba, near Fisher River First Nation (14U 613884

570051) using a harp trap. I confirmed species and sex and scanned each bat for a passive transponder (PIT tag) to rule out recaptures, at the capture site, and then placed bats in small cloth bags within a stainless-steel metal cage (20cm x 20cm x 20cm). I then transported the bats 13 km by car to a field laboratory at the Manitoba Sustainable

Development, Forestry Branch Bunkhouse. At the field lab, I weighed each bat and measured forearm length to quantify body condition. Bats that weighed less than 7.5 grams at any point of the trial, or bats that lost 5% or more of their body mass within 24 hours were released early at the capture site and not used in the study.

I used temperature data-loggers (0.5°C resolution, iButtons, DS1922L, Maxim

Integrated Products Inc., Dallas, TX, USA) to quantify torpor expression. I miniaturized data-loggers following Reeder et al. (2012) by removing the metal casings and trimming the circuit board to reduce mass. I then coated each iButton with Plasti dip (Plasti-dip

Aerosol spray, Blaine, MN, USA) and labelled each with a unique number, allowing me to use them to identify individual bats. I programmed the iButtons to record temperature every 5 minutes and attached one to each bat between the scapula using non-toxic surgical adhesive (294-Osto-bond, Montreal, Canada). Bats were held in captivity for less than 48 hours, so I glued the iButtons to the fur, rather than directly to the skin, to facilitate removal prior to release (Chapter 2). At the end of each trial, I removed iButtons by clipping the fur and downloaded Tfur data using One wire software (Maxim Integrated,

San Jose, California, USA).

For each 24-hour trial, I housed bats in an outdoor flight enclosure, large enough for bats to fly freely and select from among multiple roosts (length: 4.3m, width: 4.3m, height: 2.0 m; Model #076-5460-2, Outbound Screen House, Outbound, Toronto,

Canada). The flight enclosure included a small table that was large enough for bats to land on which was provisioned with a dish of water and a dish of 100 mealworms

(provided ad libitum, 10 per bat) gut-loaded with beta-carotene multivitamins (SRP

00300, Herptivite, CA, USA) and a nutrient supplement (Repashy Superfoods, Repashy

Ventures Inc., CA, USA) and water). I set up four roost boxes (33cm x 30cm x 3cm), one in each corner of the enclosure. Roost boxes were open at the bottom to allow bats to freely enter and exit. One of the roost boxes was insulated (Model # 547658, CodeBord

Extruded Polystyrene Rigid Insulation, 2cm thick, Foamular, Owens Corning

Commercial Insulation, Toledo, OH, USA) and heated using an outdoor thermostat

(ETC-141000-000, Ranco ETC, NEMA 4X, Delphos, OH, USA) and heating mat (Model

# PT2035, Heat Wave Desert, Exo Terra, Montreal, Canada) adhered to the back-interior wall of the roost box while the three remaining roosts were un-heated (hereafter referred to as “control”). The thermostat was set to 30°C and I recorded Troost inside each roost box using an un-modified iButton set to record every 10 min. Due to fluctuations in outside Ta, the actual Troost varied from 13.0 to 32.0°C compared to 9.5 to 28.0°C in the un-heated control roosts. I quantified daily average Troost as the mean of all 144 10-min temperature readings for each 24-h experimental trial. Heated roost boxes were significantly warmer (22.2±1.54°C) compared to control roost boxes (16.51±1.61°C,

Welch two sample T-test, t=13.3, df=24.1, p=<0.0001, Figure 3.1). Thus, my

73 experimental setups allowed me to provide bats with multiple roost selection options while simulating a rare, high quality roost among a number of lower quality roosts.

Figure 3.1: Boxplots of daily average Troost in both artificially heated and control roost boxes over the course of 11 24-hour trials (p<0.0001). Centre lines represent median values, boxes represent the interquartile range, the upper whiskers represent the third quartile plus 1.5 times the interquartile range, and the lower whiskers represent the first quartile minus 1.5 times the interquartile range. Open circles represent outliers.

To quantify pathogen transmission, I used a non-toxic ultraviolet (UV) fluorescent powder (AX-18N, Signal Green, Day-Glo Color Corp. Cleveland, Ohio, USA) to “infect” one randomly selected individual from the 10 bats captured for each trial. On the second night after capture I used a 5 mm paint brush to apply a 40 mg dose of UV powder to the dorsal side of each wing of the “infected” individual. I then immediately released the nine other “un-infected” bats into the flight enclosure with the “infected” individual. That individual was then free to interact with, and potentially transmit the proxy pathogen to, the other nine bats in the enclosure for the next 24 hours.

Twenty-four hours after introducing the proxy pathogen, I re-entered the enclosure to record Troost for each box, group size in each box, and the location of each individual. I then recaptured each bat and photographed the dorsal and ventral wing membranes under UV light (395nM Blacklight, Ledwholesalers Inc., Hayward, CA) using a digital camera (Digital Rebel Xti, Canon Inc., Oita, Japan). I weighed each bat and any individuals that were within 90% of their original starting weight were implanted with a uniquely coded passive transponder (PIT tag, Trovan Ltd. ID 100-01 Douglas,

UK) under the skin between the scapulae. This PIT tag, along with trimmed fur on the dorsum, would have allowed me to identify, and exclude recaptured individuals from subsequent trials although I never recaptured any bats over the course of the study. At the end of each trial, I released all bats at the original capture site.

After each trial, all surfaces of the enclosure were sanitized and cleaned for residual UV powder with a 10% bleach solution and all roost boxes were soaked in 10% bleach solutions (sodium hypochlorite, The Clorox Company, Oakland, CA) for 20

76 minutes. The enclosure and roost boxes were allowed to air dry for 20 hours before being used in the next trial.

Quantifying when animals are “in” versus “out of” torpor is controversial

(Barclay et al. 2001, Willis & Brigham 2003, Boyles et al. 2011, Brigham et al. 2011).

One approach is to quantify “degree-minutes,” or the area under the curve for a time course of Tsk below some Tsk threshold assumed to reflect the boundary between torpor and normothermia (Barclay et al. 2001, Lausen & Barclay 2003). Degree-minutes has limitations (Willis & Brigham 2003, Brigham et al. 2011, see Chapter 2) but for my study, I was interested only in quantifying relative variation in torpor use among individuals. Therefore, I used a method similar to that of Lausen & Barclay (2003) and calculated the area between Tsk and 0° for each bat over the 24-hour trial. I first interpolated the temperature between 5-minute readings by multiplying each Tsk reading by five. I then simply summed these measured and interpolated Tsk values for each individual over the course of each trial. I used these values as, what I term, a relative

Index of Normothermy (hereafter NI) to compare torpor expression by individuals in my study (Barclay et al. 2001, Lausen & Barclay 2003, Chapter 2). Low values of NI reflect individuals that expression deep and/or long bouts of torpor.

I quantified intensity of infection with UV powder using ImageJ (ImageJ v.

1.8.0_66, U.S. National Institutes of Health, Bethesda, Maryland, USA). I scaled images using a standardized object (i.e. U.S. penny, diameter = 19.05mm) and quantified the total area of each bat’s wing and the area covered with proxy pathogen (Fuller et al.

2011). I then calculated intensity of infection as the proportion of wing covered with UV powder (Webber 2016, Fuller et al. 2011). For bats without detectable infections (n = 3), I

77 replaced the values of zero with the lowest non-zero proportion I found for other bats divided by two, assuming that my lowest detected value approximated a minimum limit of detection (Croghan & Egehy 2003).

I used R (version 3.2.3, GUI 1.51, R Development Core Team 2015) for all statistical analyses. To determine the effect of roost box type (i.e., heated or control) on aggregation I used a Chi-squared test with expected values calculated based on the null assumption that bats would select roosts at random (i.e., a probability of being found in any box = 0.25). To assess the effect of average Troost on aggregation, I used a generalized linear model (GLM, family=binomial) with proportion of bats as my response variable and average Troost as my predictor variables. Numbers of bats fluctuated slightly among trials (i.e., from n = 6 to 10 bats per trial) because of premature releases due to mass loss and two escape events so I used proportion rather than number of bats in each roost box, as my response variable for this analysis and used a logit transformation to achieve normality.

To understand factors influencing torpor expression, I used a linear mixed effects

(LME) model with Restricted Maximum Likelihood (REML) with each individual’s NI value as the response variable and average daily Troost, group size (i.e., the number of bats found in the individual’s roost box), mass, sex, and an interaction between Troost and group size as predictor variables. Despite the differences in number of bats used for each trial, I used number of bats in a given roost box, instead of proportion of bats, as the predictor variable for this analysis based on the assumption that absolute number of bats would be the more important driver of social thermoregulation. I included an interaction between Troost and group size based on the assumption that effects of group size on torpor

78 expression could vary with Troost. I did not include Ta outside of roost boxes in my analysis because Ta significantly affected Troost for both heated and control roost boxes

(LME with trial as a random effect (p<0.001), heated roosts: t=11.4, df=9, p<0.0001, r2=0.93; unheated roosts: t=13.7, df=9, p<0.0001, r2=0.93, Figure 3.2). I also included individual trial number as a random effect to account for pseudoreplication (Dingemanse

& Dochtermann 2013).

To understand how group size and torpor expression affected pathogen transmission, I used a LME with REML to test for effects of NI, group size (i.e., number of bats found in the individual’s roost box), and sex on each individual’s intensity of infection with UV powder. I again included trial as a random effect to account for pseudoreplication and used number instead of proportion of bats based on the assumption that, as for torpor expression, the number of roost-mates was more likely to influence pathogen transmission. Intensity of infection fit a negative binomial distribution (see results) so I used a logit-transformation to achieve normality. I examined residuals to ensure that all models met LME assumptions. All values are reported as the mean ± SD and I assessed significance at α=0.05.

Results

Canada 2016). I caught 46 female and 22 male little brown bats over the course of 11 capture events. For all but one of the 11 trials, most bats roosted in the heated roost box

(Figure 3.3). On average, 76.5±2.2% of bats roosted in the heated box each day over the control boxes, which is significantly greater than the expected 25% (Χ2=169.6, d.f.=1, p<0.0001; Figure 3.3). The effect of average Troost on proportion of bats in each roost approached significance (r2=0.79, z=1.8, p=0.06, Figure 3.4) after controlling for trial as a random effect (p=0.01).

Figure 3.3: Proportions of bats roosting in one heated versus three un-heated control roost boxes over the course of 11 24-h trials.

Values of NI ranged from 46400 degree-minutes (i.e., the lowest level of torpor expression) to 37710 degree-minutes with an average of 43390±2144.417 degree- minutes. NI was significantly affected by sex, with females defending higher NI than

2 males, and an interaction between group size and Troost (r =0.88, Table 3.1). Group size fit a tri-modal distribution clearly dividing into small (1-3 bats), medium (4-6 bats), and large (7-10 bats) group sizes (Figure 3.5) so to understand this interaction I analysed the relationship between NI and Troost for each of the three group sizes independently. For small groups Troost significantly affected NI such that a 1°C increase in average Troost increased an individual’s NI by 802 degree-minutes (r2=0.95, Table 3.2). The NI incorporates both the depth and duration of torpor bouts into one unit so that an 802 degree-minute increase could represent many different responses (e.g., a 3°C increase in

Tfur lasting about 4.5 hours or a 14°C increase in Tfur lasting about 1 hour, Table 3.2).

There was no effect of sex on NI for small groups but, interestingly, the effect of mass approached significance (Table 3.2) with higher values of NI for lighter bats. Average

Troost also significantly affected individual NI for bats in large groups such that a 394 degree-minute increase in NI could represent many possible responses (e.g. a 1°C

2 increase in Tfur lasting 6.5 h or a 3°C increase lasing about 2 hours, r =0.47, Table 3.3). In large groups there was a significant sex effect with females defending NIs 857 degree- minutes warmer, on average, than males (Table 3.3). There was no effect of body mass on

NI for individuals in large groups. None of the predictor variables significantly affected

NI for medium-sized groups (r2=0.06, Table 3.4)

24-hour trials.

Variable Coefficient Standard Error t-value Degrees of p-value Freedom Group size 1493 286 5.23 53 <0.0001 Average 930 85.9 10.8 53 <0.0001 Troost Group size: -66.5 13.4 -4.96 53 <0.0001 Average Troost Sex -169 217 -2.17 53 0.035 Mass 120 62.3 1.93 53 0.059

Figure 3.5: Histogram of the group size (number of bats in roost) in which each of the 68 little brown bats chose to roost over 11 24-hour trials (see results).

Figure 3.6: Scatter plot showing the effect of average Troost on normothermy index.

Group size showed a tri-modal pattern (see Figure 3.4) and is represented here by black circles for groups of 1-4 individuals, dark gray circles for medium-sized groups of 4-7 individuals, and white circles for large groups of 8-10 individuals. Relationships between groups size and NI were significant for small and large but not medium-sized groups (see results).

Standard Degrees of Variable Coefficient t-value p-value Error Freedom Average 802 47.0 18.1 9 <0.0001 Troost Sex -153 169 -0.90 9 0.39 Mass -177 82.4 2.15 9 0.06

Standard Degrees of Variable Coefficient t-value p-value Error Freedom Average 394 86.1 4.58 3 0.020 Troost Sex -1006 294 -3.43 28 0.002 Mass 89.6 120 0.74 28 0.46

Standard Degrees of Variable Coefficient t-value p-value Error Freedom Average -104 344 -0.303 1 0.81 Troost Sex -117 599 -0.196 9 0.85 Mass -126 167 -0.756 9 0.47

Three individuals had no proxy pathogen on their wings while all others had at least some infection for an overall prevalence of 95.5%. The greatest intensity of infection (proportion of wing covered by UV powder) was 11.8% with an average of

0.96±0.02%. Infection intensity showed a negative binomial distribution with few individuals having high infection intensities and most individuals having low infection intensities (Figure 3.7a), but I achieved a normal distribution using a logit transformation

(Figure 3.7b, Shapiro-Wilk normality test, W=0.98, p=0.45). None of NI, group size or sex had any effect on infection intensity (r2=0.06, Table 3.5, Figure 3.8 and 3.9).

Variable t-value Degrees of Freedom p-value Group size 0.255 54 0.800 NI -0.409 54 0.684

Interaction between group -0.208 54 0.836 size and NI

Sex -1.606 54 0.114

A B

Figure 3.8: Scatter plot of the relationship between logit-transformed intensity of infection for each individual and the size of the group in which the bat was found

(p=0.80).

Figure 3.9: Scatter plot of the relationship between logit-transformed intensity of infection for each individual and it’s NI (p=0.68).

Discussion

My results provide the first experimental evidence that warm roosting habitat affects both aggregation sizes and torpor expression in a colonial, temperate-zone bat.

However, I found no evidence that either aggregation size or torpor expression influenced intensity of infection with a proxy pathogen. Taken together, these results lend support to

Wilcox and Willis’ (2016) habitat enhancement hypothesis that providing bats with relatively warm roosting habitat during the active season could help them reduce torpor expression and potentially improve rates of healing and recovery from WNS, as well as speed rates of reproduction and offspring growth.

Consistent with my first hypothesis, bats were disproportionately more likely to roost in the one roost box in their enclosure that was heated compared to un-heated control roost boxes. There was also a trend for a positive effect of Troost on aggregation size that approached significance. Willis and Brigham (2007) found a similar positive relationship between group size and Troost for big brown bats (Eptesicus fuscus) but in their study, group size appeared to drive Troost and not the other way around (i.e., more bats in a given tree hollow caused warmer Troost). Because my heated boxes were artificially heated and were consistently warmer than control boxes throughout all trials, independent of group size, bats likely had little to no effect on Troost.

In support of my second hypothesis, bats that roosted in warmer roosts and with more conspecifics used less torpor. Larger numbers of bats tended to aggregate in heated roosts and those bats employed less torpor. In small groups, Troost had a strong effect on

NI which suggests that bats inclined to use torpor may select colder roosts and avoid large groups of conspecifics. There was also a trend (p=0.06) for higher NI in lighter bats

96 than heavier bats in small groups in my experiment. Stawski and Geiser (2010) also found similar trends, in that bats in better body condition used more torpor than those in poor condition. They hypothesized that this pattern may be because bats are employing torpor as a means of reducing activity to avoid predation, not strictly as an energy-saving mechanism. There was also a positive effect of Troost on NI for bats in large groups, but this effect was less pronounced than for small groups suggesting that a combination of passive aggregation in warm roosts, plus reduced heat loss from social thermoregulation, allows bats in larger groups to avoid torpor expression (Figure 3.6). Interestingly, for large groups, there was also an effect of sex on torpor expression with females defending higher NI than males. If anything, I expected that males should have defended higher Tfur than females at the time of year of my study because of the importance of warm Tb for sperm production. One possibility is that females, which are highly social during summer, spent more time out of torpor while in large groups in my experiment because of behaviours associated with social interactions between conspecifics.

Communal roosting can theoretically increase pathogen transmission (Alexander

1974). Although Troost affected bat aggregation size and torpor expression, I found no support for my third hypothesis that group size or torpor affect pathogen transmission in this species, despite the fact that transmission dynamics of my proxy pathogen approximated that for most natural pathogens with most individuals exhibiting low intensity of infection and only a few individuals being heavily infected (e.g., Shaw et al.

2016). At least at this small group size of a maximum of 10 bats, and over a 24-hour time scale, neither aggregation nor torpor expression affected pathogen transmission. This pattern could reflect the influence of host traits other than aggregation and torpor

97 expression. Hillegass et al. (2008) found similar results in Cape ground squirrels in that group size had no effect on ectoparasite or endoparasite loads, but instead sex was a more important determinant of parasite loads. Males had 3 times as many ectoparasites as females, while females had 3 times as many endoparasites. Hillegass et al. (2008) suggested that this sex difference may be caused by sexual selection, in that females have smaller home ranges (greater likelihood of coming into contact with endoparasites) and allogroom more frequently (reducing ectoparasite load) compared to their male conspecifics that have greater ectoparasite loads possibly because of high androgen levels and lower grooming times. However, I did not find an effect of sex on pathogen acquisition in my study. Parasite transmission may also vary with differences in the mobility of the host and the mode of parasite transmission (Patterson & Ruckstuhl 2013).

A meta-analysis conducted by Patterson and Ruckstuhl (2013) found that group size does have a significant positive effect on pathogen transmission overall, but when focusing only on mobile parasites, pathogen load is negatively correlated with group size and when investigating only mobile hosts, group size does not affect pathogen load either. In the case of my study, the proxy pathogen was transmitted via direct contact and could be considered density dependent, so in larger groups pathogen load should have been greater.

Using captive bats allowed me to control for a range of variables, and also recapture individuals to assess intensity of infection after a consistent time interval.

However, captivity and my experimental design exposed bats to conditions that would differ, potentially dramatically, from those experienced by free-ranging bats. For one, bats in my study were provided food ad libitum, and the available food was never

98 completely consumed. Thus, bats may have been in positive energy balance more often than would occur in the wild, which could have influenced roost preferences and torpor expression. If bats had limited food availability they might have been motivated to conserve energy, select colder roosts and express torpor (e.g., Lovegrove et al. 2001,

Mzilikazi & Lovegrove 2002). On the other hand, Ta during my study rarely fell below 11

°C during the first few hours after sunset (when most bat foraging activity occurs). Given the strong relationship between Ta and flying insect availability, free-ranging bats would probably only rarely have been limited by food availability on most of the nights when I conducted my experiment. Manipulating Ta and food availability in future studies could provide additional insight into factors affecting bats’ thermoregulatory behaviours in the wild.

In addition to ad libitum food, the spatial and temporal scale of my experiment was small relative to natural home range size for little brown bats which could have influenced my ability to quantify pathogen dynamics. The results of my power analysis show that in order to detect a large effect of NI, group size, sex, and mass on pathogen acquisition, my sample size would have had to be 131 bats, compared to my 68 individuals. Based on an a priori power analysis, I recommend future studies conduct 15-

20 trials of 10 bats per trial, in order to detect large, biologically significant results.

However, because of the life history of bats, I would not have been able to capture bats earlier in the summer or later in the fall for my experiment. Each trial had a maximum of

10 bats and the median roosting group size I observed was 8 individuals per roost box, which is relatively small compared to maternity colony sizes (Davis & Hitchcock 1965,

Barclay & Cash 1985, Kalcounis & Brigham 1994, although smaller group sizes could be

99 more likely during other times of year). This behaviour may have reduced the potential for interactions among bats in my study and caused me to underestimate pathogen transmission relative to natural colonies. Bats inside each roost were considered to be part of one group, but did not necessarily cuddle in a tightly packed group. On the other hand, the small spatial scale available to bats in my experiment could have increased the likelihood of interactions between conspecifics, in turn increasing the chance of pathogen transmission. In this case, high contact rates in my flight enclosure may have effectively swamped any signal of torpor expression or aggregation size on pathogen transmission. If this was the case, however, I would have expected very high intensities of infection for most bats and which is not what I observed. The negative binomial distribution of infection intensity I observed is consistent with patterns seen for most pathogens and contact parasites in nature and suggests that transmission dynamics of my proxy pathogen approximated those of a natural pathogen (Shaw et al. 2016). Pathogen loads could have also been altered as bats groomed off the powder as it was observed in their feces.

Although such studies would be challenging, I recommend future attempts to use larger group sizes of species that are more tractable in captivity (e.g., big brown bats) or free- ranging bats roosting in different contexts (e.g., rock crevices vs. buildings, Lausen &

Barclay 2006). Studies of free-ranging bats could potentially quantify prevalence and/or loads of naturally occurring pathogens like the rocky mountain coronavirus in big brown bats (Dominguez et al. 2007, Misra et al. 2009) or the Myotis coronavirus (Misra et al.

2009) which appear to chronically infect colonial bats with little pathology and no evidence of risk for humans. Using observational studies of existing viruses, combined with experiments like mine, could help improve understanding of the influence of

100 roosting aggregation and torpor expression on pathogen dynamics in the wild.

My study provides further evidence that summer habitat enhancement could be a valuable management tool for conservation of bat species at risk from WNS and other threats. For some bat species that regularly use anthropogenic structures (e.g., little brown bats), artificial heating of roost boxes could be a viable management option. Other endangered species, however, are less likely not use anthropogenic structures and, instead, rely much more heavily on tree hollows and other roosts in forests (e.g., northern long-eared bats). For these species, identifying and protecting habitat features that naturally increase Troost may also be important for management. Older, larger trees on south-facing slopes may provide relatively warm microclimates and my results suggest that other habitat features which enhance warm microclimates should be identified and preserved. Protecting and enhancing summer roosting habitat could be a viable management option for conserving bats species at risk.

Taken together, my results lend support to the habitat enhancement hypothesis, that managing bat habitats in ways that increase availability of warm roosts, could be beneficial for helping bat populations recover from WNS and other threats (Wilcox and

Willis 2016). Reducing torpor expression could help bats heal and recover more quickly from WNS in spring (Chapter 2, Faure et al. 2009). After recovery, warm roosts may also improve reproductive success of WNS survivors by speeding fetal growth, enhancing lactation and growth rates of juveniles (e.g. McCarty & Winkler 1999), and enhancing sperm production for males in summer and fall (Jagiello et al. 1992). Although bats in my study were all WNS-negative, like control bats studied by Wilcox and Willis (2016), they still showed a strong preference for warm roosts and spent more time at warmer Tb when

101 roosting in warm structures. In addition, I found no evidence for an increase in pathogen or parasite transmission for bats using less torpor in heated roosts, which is one potential downside of habitat enhancement. More work is needed with free-ranging bats, but so far, habitat enhancement appears to have promise as a potential management strategy for bats imperiled by WNS and other threats.

102

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CHAPTER 4: GENERAL CONCLUSIONS

Pathogens of wildlife can affect host survival and reproductive success, which may lead to species declines or even extinctions and loss of overall biodiversity (Daszak et al. 2000, Smith et al. 2006, Mitchell et al. 2008). Host behaviour, like aggregation, can affect an individual’s overall pathogen load. Larger aggregations of hosts should have more interactions, which could result in greater pathogen transmission (Altizer et al.

2003). High quality habitat, when surrounded by lower-quality habitat, can cause aggregations of conspecifics. These aggregations can bring about many benefits, (e.g., increased predator vigilance and decreased thermoregulatory costs, however, they could also bring about detrimental costs (i.e. greater likelihood of acquiring pathogens.

White-nose syndrome (WNS) has caused drastic population crashes, resulting in regional declines and anticipated regional or global extinction of several species of hibernating bats (Frick et al. 2010, Langwig et al. 2012). Providing bats with warm microclimates, either by preserving or enhancing existing habitat or by creating artificial habitat, may help survivors recovering from WNS heal more quickly in spring and reproduce faster in summer (Wilcox & Willis 2016). Therefore, warm summer roosts could help populations recover from WNS as long as aggregation at these warm roosts do not increase transmission of P. destructans or other harmful pathogens. I tested the over- arching hypothesis that bats prefer roosting in warm microclimates, which enables them to reduce the energetic cost of defending warm body temperatures (Tb) without entering torpor. I also investigated whether the lesser use of torpor may affect their likelihood of acquiring pathogens if it caused them to come into contact with more infected individuals or substrates. My research showed that bats preferred warm roosts and that warm roosts

111 reduced their use of torpor (potentially allowing them allocate energy to other processes like healing or growth), with no evidence of increased rates of pathogen acquisition.

In Chapter 2, I examined the relationship between habitat quality and torpor use and tested how torpor use could affect the likelihood of acquiring substrate-transmitted pathogen. I tested whether warm roosting habitat would reduce the amount of torpor used by bats. I predicted that bats that had access to warm, artificially-heated roost boxes would employ less torpor than bats with access to only un-heated control roost boxes because of the energetic benefits gained from warm microclimates. Second, tested whether the reduced torpor use would increase the likelihood of acquiring a contact pathogen from an infected substrate, likely because of increased activity. I predicted that bats provided with warm roosts would show greater prevalence and intensity of infection.

I found a positive relationship between habitat quality and torpor expression with individuals using less torpor in warm roosts. At least on the small spatial and temporal scale of my experiment, I found no effect of torpor expression or roost temperature in intensity of infection with ultraviolet fluorescent powder applied to substrates in the experimental environment. From a management perspective, these results suggest that providing artificial high-quality roosts may help survivors of WNS recover and reproduce after emerging from hibernation without increased risk of acquiring pathogens or parasites from infected substrates.

In Chapter 3, I examined the relationship between habitat quality and aggregation, and how they affect torpor expression. I then examined how aggregation and torpor expression affect pathogen transmission. First, I tested whether limited, warm roosts would increase passive aggregation by groups of bats. I predicted that if groups were

112 given access to limited, warm roosting habitat, that the group size of aggregations would increase. Second, I tested whether limited, warm roosting habitat and/or large aggregations would affect thermoregulation. I predicted that bats in warm roosts and/or with larger groups would use less torpor than those in colder roosts or in smaller groups.

Third, I tested whether aggregation and/or torpor use would increase the risk of pathogen acquisition. I predicted that bats roosting in larger groups and/or using less torpor would have greater prevalence and intensity of infection after an infected individual was introduced.

I found that bats disproportionately chose to aggregate in warm roosts. I also found a negative relationship between habitat quality (Troost), group size, and the interaction between Troost and group size, and the normothermy index of bats. As group size increased, the effect of Troost on torpor use decreased. Overall, the greater Troost and the larger the aggregation, the less torpor the bat used, which also follows the predictions of the social aggregation hypothesis. Endotherms that social thermoregulate may benefit from huddling and warm microclimates to the point of reducing the need to enter torpor.

My research has important implications for conservation and wildlife management. My results suggest that bats currently recovering from WNS, and which use anthropogenic structures may benefit from heated roosts, to reduce the cost of maintaining higher Tb, allowing them to invest more into healing, reproduction, and offspring development. Enhancement to forest roosting habitat could also be important for species which do not use artificial structures and my results suggest that significant effort should be undertaken to understand habitat features of forest roosts that might increase roost temperatures. If habitat enhancement can help animals reduce

113 thermoenergetic costs, then it could help them to invest more energy into recovering from disease, reproducing, and raising young, leading to lower death rates and greater population growth rates. Encouraging the survival and reproduction of WNS survivors should be a conservation priority as evolutionary rescue may be the most viable management option to avoid extinction of bats affected by WNS.

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References

Daszak, P., Cunningham, A.A., & Hyatt, A.D. 2000 Emerging infectious diseases of wildlife- threats to biodiversity and Human Health. Science 287, 443-448

Mitchell, K.M., Churcher, T.S., Garner, T.W.J. & Fisher, M.C. 2008 Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction Proceedings of the Royal Society B. 275, 329-334.

Smith, K.F., Sax, D.F., & Lafferty, K.D. 2006 Evidence for the role of infectious disease in species extinction and endangerment Conservation Biology 20, 1349-1357

Wilcox, A. & Willis, C.K.R. 2016 Energetic benefits of enhanced summer roosting habitat for little brown bats (Myotis lucifugus) recovering from white-nose syndrome. Conservation physiology 4, 1-12.

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